Curtobacterium pruni sp. nov. causing black blotch on red apricot fruit in China

doi:10.21203/rs.3.rs-7415476/v1

Download PDF

Research Article

Curtobacterium pruni sp. nov. causing black blotch on red apricot fruit in China

https://doi.org/10.21203/rs.3.rs-7415476/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

The red apricot (Prunus armeniaca ‘Bei-zhai-hong-xing’) is exclusively cultivated in Beijing and highly popular in the Chinese market due to its nutrient-rich fruits, which contain vitamin C, carotene, calcium, phosphorus, and potassium. In May 2021, distinct black spots or black botches were observed on fruits in many orchards and approximately 50% of fruits were infected, resulting in significant economic losses for farmers. Literature review confirmed no prior identification of the causal pathogen. Early-stage symptoms manifested as small, brown circular lesions that rapidly enlarged and turned black and formed black blotches within several days; no mycelia or spores were observed. Fruits exhibited high susceptibility, whereas leaves remained asymptomatic. Single colonies were isolated from diseased fruits. Representative isolate of Hongxing underwent pathogenicity testing and identification via morphological characterization and 16S rRNA gene phylogenetic analysis. Cells of the pathogen were rod-shaped, Gram-stain-positive, non-spore-forming and of (0.5-3.0) µm × (0.2–0.6) µm in size. The whole-genome sequencing of strain Hongxing showed that the circular chromosome comprised 3,884,414 bp with 70.98% G + C content and encoded 3,657 protein-coding genes. The phylogenomic comparison with other Curtobacterium species demonstrated that the strain Hongxing is distinctive from other known Curtobacterium species. Combined morphological and phylogenetic analyses identified strain Hongxing as a novel Curtobacterium species pathogenic to apricot fruit. We propose the novel species Curtobacterium pruni sp. nov., with type strain Hongxing^T. To the best of our knowledge, this is the first report of Curtobacterium sp. as a pathogen on red apricot fruits.

Curtobacterium pruni sp.nov.

Prunus armeniaca

phylogeny

genome sequence

Most Curtobacterium species are phytopathogens, causing diseases such as bacterial wilt of common bean (Wendland et al., 2016). However, certain strains of C. flaccumfaciens act as beneficial microbes that promote plant growth (Behrendt et al., 2002; Chase et al., 2017) and suppress plant diseases (Lacava et al., 2007). Curtobacterium flaccumfaciens pv. flaccumfaciens is a major pathogen of legumes, including common bean, soybean, and cowpea (Osdaghi, 2014; Harveson et al., 2015; Wendland et al., 2016; Sammer & Reiher, 2012). Bacterial wilt management is challenging due to the seedborne nature of Curtobacterium spp. and their persistence on weeds and crop debris in the field (Osdaghi et al., 2020; Nascimento et al., 2020). Recently described pathogens include Curtobacterium allii, the causal agent of onion bulb rot (Khanal et al., 2022). Rare human infections linked to Curtobacterium strains have also been documented (Francis et al., 2011), suggesting potential alternative hosts beyond plants.

The red apricot (Prunus armeniaca ‘Bei-zhai-hong-xing’) is highly popular in China for its thin-skinned, thick-fleshed fruits with small pits and high nutrient content (e.g., vitamin C, carotene, fructose, organic acids, protein, calcium, phosphorus, potassium). This cultivar is exclusively cultivated in Beijing’s Pinggu District due to specific geographic requirements and provides significant income for local farmers. In May 2020, distinct black blotches were observed on fruits in multiple orchards across Pinggu District, Beijing. Initial symptoms appeared as small, brown circular lesions that rapidly enlarged and blackened within days; no mycelia or spores were observed. Fruits exhibited high susceptibility, whereas leaves remained asymptomatic. Other local apricot cultivars showed lower infection rates. Approximately 50% of fruits in affected orchards were infected, causing substantial economic losses. A literature review indicated the causal agent was unidentified. This study characterizes the pathogen and establishes the taxonomic status of the causal pathogen.

Field samplings and pathogen isolation

Diseased apricot fruits (Prunus armeniaca ‘Bei-zhai-hong-xing’) were collected from orchards in Pinggu District, Beijing, China (40°01′N, 117°01′E). Five symptomatic fruits displaying typical black blotches were randomly sampled. Pathogen isolation followed Guarnaccia et al. (2021) with minor modifications. Symptomatic tissues (0.5–1.0 cm) from lesion margins were surface-disinfected in 70% ethanol (30 s), 1% sodium hypochlorite (30 s), rinsed in sterile distilled water, dried on sterile filter paper, and plated on Luria-Bertani agar (LBA). Plates were incubated at 25 ± 1°C for 48 h. Single colonies were re-streaked onto fresh LBA plates. After 48 h incubation, single colonies were re-isolated on LBA to obtain pure cultures. Twenty isolates were obtained; two (Hongxing and HX-A) were selected for molecular characterization (Table 1). Stock cultures were stored on LBA slants at 4°C for further study.

Test of pathogenicity

The pathogenicity was verified according to the procedures by Ogiso et al. (2002) with minor modifications. A bacterial suspension (10⁴ CFU/mL) from pure cultures of the pathogen prepared from LB was sprayed onto five healthy apricot fruits (Prunus armeniaca “Bei-zhai-hong-xing” ) in the orchard. The fruits inoculated with LB diluted by 10×fold with sterilized distilled water served as controls. Symptoms were recorded 7 days post-inoculation.

Morphological Characterization

Morphological characterization of the pathogen strains was carried out according to the methods described by Khanal et ac., (2022), with minor modifications.The pathogen strain was preliminarily identified by using morphological characteristics. The strain was inoculated on an LBA medium and observed for culture characters after incubation at 25°C for 48h. The strain cultured for 18–24 h on LBA plates was subjected to gram staining. Cell morphology was observed and photographed using an Olympus BX51 microscope. Biochemical tests were performed by using the API 50 CH Identification Kit. All the experiments were evaluated in triplicate to obtain accurate data.

Molecular and Phylogenetic analysis

The pathogen strain was further identified through the analysis of its 16S rRNA gene sequences. Briefly, the DNA of the strain was extracted by using the TIANamp bacterial DNA extraction kit and stored at 4°C. The 16S rDNA was amplified by polymerase chain reaction (PCR) with the bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction mixture contained 1 µL of DNA template, 5 µL of 10× PCR buffer, 4 µL of dNTPs (2.5 mmol/L), 3 µL of MgCl2 (25 mmol/L), 1 µLof each primer (1 mmol/L), 0.5 µL of Taq polymerase and 34.5 µL of ultrapure water. The reaction condition of 16S rDNA amplification was as follows: 94°C for 5 min; 30 cycles of 94°C for 1 min, 55°C for 30 s and 72°C for 1 min; and a final extension at 72°C for 10 min. Amplicons were electrophoresed on 1% agarose gels and sequenced (Beijing Tianyihuiyuan Biotechnology Co., Ltd.). DNA sequence homology searches were performed by using the online BLAST search engine in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were constructed from 16S rRNA sequences using MEGA7 (neighbor-joining method).

Genome sequencing and genome annotation

Genomic DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega) according to manufacture’s protocol. Purified genomic DNA was quantified by TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High quality DNA (OD260/280 = 1.8 ~ 2.0, >1µg) was used to do further research.

For Illumina sequencing, at least 1µg genomic DNA was used for each strain in sequencing library construction. DNA was sheared (400–500 bp) using a Covaris M220 (per manufacturer’s protocol).. Illumina sequencing libraries were prepared from the sheared fragments using the NEXTflex™ Rapid DNA-Seq Kit. Briefly, 5’ prime ends were first end-repaired and phosphorylated. Next, the 3’ ends were A-tailed and ligated to sequencing adapters. The third step is to enrich the adapters-ligated products using PCR. The prepared libraries then were used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina HiSeq X Ten machine. Bioinformatics analyses used Majorbio Cloud Platform (www.majorbio.com).

The original image data is transferred into sequence data via base calling, which is defined as raw data or raw reads and saved as FASTQ file. Those FASTQ files are the original data provided for users, and they include the detailed read sequences and the read quality information. A statistic of quality information was applied for quality trimming, by which the low quality data can be removed to form clean data. An assembly of the clean reads were performed using SOAPdenovo2 (Koren et al., 2017).

Glimmer (Delcher et al., 2007) was used for CDS prediction, tRNA-scan-SE (Borodovsky et al., 1993) was used for tRNA prediction and Barrnap was used for rRNA prediction. The predicted CDSs were annotated from NR, Swiss-Prot, Pfam, GO, COG and KEGG database using sequence alignment tools such as BLASTP, Diamond and HMMER. Briefly, each set of query proteins were aligned with the databases, and annotations of best-matched subjects (e-value < 10^− 5) were obtained for gene annotation.

The phylogeny based on the whole genome data was determined by calculating the average nucleotide identity with closely related Curtobacterium species with the Orthologous Average Nucleotide Identity Tool (OrthoANI)(Lee et al., 2016). Genome-based taxonomy was analyzed using TYGS (https://tygs.dsmz.de; Meier-Kolthoff & Göker, 2019).

Field survey and pathogen isolation

Most of apricot fruits were infected by Curtobacterium sp., and showed the typical black blotches (Fig. 1). Diseased fruits were collected and totally 20 isolates were obtained and showed the same morphology of colones on LBA plates (Fig. 2). Two pure cultures, respectively designated Hongxing and HX-A, were selected for further analysis (Table 1).

Table 1
Two typical strains of the pathogen isolated from the diseased red apricot fruits
Strains	Color and shapes of colonies	GenBank NO. of 16s rRNA gene
Hongxing^T	Yellow, round, and smooth surface	OP721100
HX-A	Yellow, round, and smooth surface	OP614835.1

Test of pathogenicity

By 7 days post-inoculation (dpi), symptoms of black sunken lesions identical to field infections appeared on inoculated fruits (Fig. 3a). At 14 days post inoculation, the typical black blotches were enlarged and the fruits become yellow in color (Fig. 3b). In this experiment, in the control groups where the the fruits were inoculated inoculated with LB diluted by 10×fold with sterilized distilled, no black blotches was observed on fruits at 7 days (Fig. 3c) or 14 days (Fig. 3d) post inoculation. The results showed that the strains used for inoculation are the pathogen which caused the black blotch on red apricots in the orchards, and the pathogenicity of the pathogen on red apricots was confirmed.

Morphological Characterization

The colony of the pathogen strain Hongxing was round or oval, ivory off-yellow and nontransparent with an smooth surface, and had a white round spot in the middle on LBA plates after 48 h of growth. Under microscope with 100× magnification, the cells of strain Hongxing was rod-shaped, Gram-stain-positive, non-spore-forming and of (0.5-3.0) µm × (0.2–0.6) µm in size (Fig. 4), the morphology characterization is consistent to the genus Curtobacterium described by Yamada and Komagata (1972).

Molecular and Phylogenetic analysis

Morphological characterizations were further confirmed by phylogenetic analysis of the sequences of 16S rRNA gene. Sequence data were deposited to the NCBI (Table 1). Comparison of 16S rRNA gene sequences with the nucleotide database of DDBJ/EMBL/GenBank using BLASTn search revealed that the strain Hongxing and strain HX-A were placed into a single clade, and are different from other known species of the genus Curtobacterium. The results of phylogeny analysis suggested that the strain Hongxing and the strain HX-A belongs to a novel species of the genus Curtobacterium (Fig. 5).

Genome sequencing and genome annotation

The genome assembly of Hongxing consisted of 1 contig larger than 1 Kbp, with a total size of 3,884,414 bp and mean GC content 70.76%. The genome of Hongxing possesses 3657 genes and encodes at least 12 rRNAs, 10 sRNA and 40 tRNAs (Table 2 and Fig. 6). The genome of the strain Hongxing also harbored genes related to cell mobility and defense mechanisms (Fig. 6 and Fig. 7). The analysis of the amino sequences of the whole genome by the software showed that the pathogen belongs to the bacteria of Type III secretion system (T3SS).

Genome sequence phylogenies showed that the strain Hongxing is closely related species of Curtobacterium flaccumfaciens CFBP 3418 and Curtobacterium flaccumfaciens LMG 3645 (type strains), respectively and that the strain Hongxing presented dDDH values of 66.2% with the latter species. The phylogeny analysis of the whole genomes of strain Hongxing and other known species of the genus Curtobacterium sp. revealed that the strains Hongxing and HX-A belonged to a novel species and formed a single clade in the phylogenetic tree (Fig. 8), suggesting that the pathogen causing black blotches on apricot fruits is a novel disease pathogen.

Table 2
General genomic features of *Curtobacterium* sp. strain Hongxing
Feature	Hongxing
Genome size (bp)	3,884,414
Number of genes	3657
Number of RNAs	62
Longest contig	3,884,414 bp
GC content (%)	70.76
N50	3,884,414 bp
N75	3,884,414 bp
L50	1
L75	1

Phylogenetic analysis of the whole genome

Genome sequence phylogenies showed that the strain Hongxing is closely related species of C. flaccumfaciens CFBP 3418 and C. aurantiacum CFBP 8819 (Fig. 8). The results of the digital DNA-DNA hybridization showed that dDDH of the genomes of strain Hongxing and C. flaccumfaciens CFBP 3418 and C. aurantiacum CFBP 8819 were 57.7% (d6) and 54.9% (d6), respectively, which were lower than 70%, confirming that the strain Hongxing was a novel species of the genus Curtobacterium (Table 3).

Table 3
The results of the digital DNA-DNA hybridization of the genomes of strain Hongxing and Curtobacterium spp.*
Query strain	Subject strain	dDDH (d0, in %)	C.I. (d0, in %)	dDDH (d4, in %)	C.I. (d4, in %)	dDDH (d6, in %)	C.I. (d6, in %)	G + C content difference (in %)
Hongxing	C. flaccumfaciens CFBP 3418	66.2	[62.4–69.9]	33.7	[31.3–36.3]	57.7	[54.5–60.9]	0.2
Hongxing	C. flaccumfaciens LMG 3645	66.2	[62.4–69.9]	33.7	[31.3–36.2]	57.7	[54.5–60.8]	0.22
Hongxing	C. aurantiacum CFBP 8819	62.7	[58.9–66.3]	33.3	[30.9–35.8]	54.9	[51.8–58.0]	0.09
Hongxing	C. allii 20TX0166	61.2	[57.5–64.8]	33.3	[30.9–35.8]	53.8	[50.7–56.9]	0.02
Hongxing	C. pusillum ATCC 19096	49.8	[46.4–53.2]	28.2	[25.9–30.7]	43.3	[40.3–46.3]	0.09
Hongxing	C. caseinilyticum GDMCC 1.2667	45.8	[42.5–49.3]	26.3	[23.9–28.7]	39.6	[36.6–42.6]	1.18
Hongxing	C. albidum DSM 20512	44	[40.6–47.4]	26.3	[23.9–28.8]	38.3	[35.4–41.4]	1.15
Hongxing	C. citreum JCM 1345	44.1	[40.7–47.5]	26.1	[23.8–28.6]	38.3	[35.4–41.4]	1.26
Hongxing	C. luteum DSM 20542	46.5	[43.1–49.9]	26.1	[23.7–28.6]	40	[37.0–43.0]	0.92
Hongxing	C. luteum JCM 1480	46.5	[43.1–49.9]	26	[23.6–28.5]	39.9	[36.9–43.0]	0.99
Hongxing	C. guangdongense RRHDQ66	45.9	[42.5–49.4]	25.8	[23.5–28.3]	39.5	[36.5–42.5]	0.22
Hongxing	C. aetherium L6-1	36.7	[33.3–40.2]	25.3	[22.9–27.7]	32.8	[29.9–35.9]	1.25
Hongxing	C. herbarum DSM 14013	37.4	[34.0–40.9]	25.2	[22.9–27.7]	33.3	[30.3–36.4]	0.66
Hongxing	C. salicis WW7	38.3	[34.9–41.8]	24.5	[22.2–27.0]	33.7	[30.7–36.8]	0.61
Hongxing	C. ammoniigenes NBRC 101786	17.6	[14.5–21.1]	20.3	[18.0–22.7]	17.3	[14.7–20.3]	2.91
Hongxing	C. plantarum LMG 16222	12.5	[9.8–15.8]	19	[16.8–21.4]	12.9	[10.6–15.7]	15.72

* The genome of strain Hongxing was subject to the website of https://tygs.dsmz.de/ for digital DNA-DNA hybridization analysis to further confirm the taxonomy of the causal pathogen.

This research described the pathogen causing novel disease of black blotches on apricot fruits in China, and based on morphology characterization and the results of the phylogenetic tree analysis16S rRNA gene sequence and the genome sequences, the pathogen was identified as a novel species of the genus Curtobacterium sp.. Summarizing the morphology and molecular characterization, the novel species Curtobacterium pruni sp. nov., was proposed. Although some species of the genus Curtobacterium have been well documented as a common plant pathogen with the potential to cause significant disease in many legumes, to the best of our knowledge, this is the first report of Curtobacterium sp. as a pathogen on red apricot fruits.

Authors' contributions

Conception and design of the study: D.P. Zhang and J.B. Liu.

Performed experiments: H.Q. Yang and R.Q. Wang.

Analyzed data: C.G. Lu, Y. Yang, L. Liu.

Wrote the manuscript: Y. Yang, P. Wu, D.P. Zhang.

All authors read and approved the final manuscript.

Acknowledgments

The authors thank the project of National Key R&D Program of China（Grant number ：2024YFD1501304）

Funding

This project was supported by National Key R&D Program of China（Grant number ：2024YFD1501304）.

Conflict of interest

The Authors declare that they have no conflict of interest.

Compliance with ethical standards

This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with human participants or animals performed by any of the authors.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Fujinaga M, Yamagishi N, Ogiso H, Takeuchi J, Moriwaki J, Sato T (2011) First report of celery stunt anthracnose caused by Colletotrichum simmondsii in Japan. J Gen Plant Pathol 77:243–247.
Guarnaccia V, Martino I, Gilardil G, Garibaldi A, Gullino ML (2021) Colletotrichum spp. causing anthracnose on ornamental plants in northern Italy. J Plant Pathol 103:127–137.
Farris JS. Estimating phylogenetic trees from distance matrices. Am Nat. 1972;106: 645–667.
Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 2019;10: 2182.
Wendland E, Nascimento RM, Ferreira LM, Souza EJ, Maccheroni W Jr (2016) First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial wilt of common bean in Brazil. Plant Dis 100:414.
Behrendt U, Munch JC, Kottke I, Berg G (2002) Endophytic bacteria isolated from roots of field-grown maize (Zea mays L.) enhance the growth of canola (Brassica napus L.) seedlings under gnotobiotic conditions. Biol Fertil Soils 36:112–117.
Chase AJ, Beattie GA, Goodfellow M (2017) Diversity and plant growth-promoting traits of culturable endophytic bacteria isolated from field-grown maize (Zea mays L.). Plant Soil 418:191–206.
Lacava PT, Azevedo JL, Maccheroni W Jr, Araújo WL, Monteiro RV (2007) Endophytic bacteria isolated from coffee plants (Coffea arabica L.) promote growth and protect against phytopathogens. Res Microbiol 158:86 - 93.
Osdaghi E, Kharazmi M, Ghorbani M (2014) First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial wilt of common bean in Iran. Plant Dis 98:437.
Harveson RM, Osborne LD, Wrather JA (2015) First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial wilt of soybean in Nebraska. Plant Dis 99:1026.
Wendland E, Nascimento RM, Ferreira LM, Souza EJ, Maccheroni W Jr (2016) First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial wilt of common bean in Brazil. Plant Dis 100:414.
Sammer U, Reiher H (2012) First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial wilt of common bean in Germany. Plant Dis 96:1821.
Osdaghi E, Kharazmi M, Mohammadi M, Safaraei-Mahroo S, Bahar M (2020) Management of Curtobacterium flaccumfaciens pv. flaccumfaciens, the causal agent of common bean bacterial wilt. Arch Phytopathol Plant Prot 53:869–884.
Nascimento RM, Lopes AR, Souza EJ, Maccheroni W Jr, Wendland E (2020) Detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean seeds by loop-mediated isothermal amplification (LAMP). Eur J Plant Pathol 156:493–502.
Khanal S, Adhikari B, Adhikari K, Shrestha R, Chhetri P, Gautam R (2022) First report of onion bulb rot caused by Curtobacterium allii in Nepal. Plant Dis 106:2404.
Francis KP, Chitnis SS, Srinivasan V, Narayanan S, Krishnan S (2011) Curtobacterium flaccumfaciens subsp. flaccumfaciens bacteremia in a patient with acute myeloid leukemia. J Clin Microbiol 49:820 - 822.
Guarnaccia V, Martino I, Gilardi G, Garibaldi A, Gullino ML (2021) Colletotrichum spp. causing anthracnose on ornamental plants in northern Italy. J Plant Pathol 103:127–137.
Khanal S, Adhikari B, Adhikari K, Shrestha R, Chhetri P, Gautam R (2022) First report of onion bulb rot caused by Curtobacterium allii in Nepal. Plant Dis 106:2404.
Koren S, Schatz MC, Walenz BP (2017) Canu: scalable and accurate long-read assembly via adaptive k -mer weighting and repeat separation. Genome Research 27:722 - 736.
Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
Lee JH, Ouk Kim Y, Park S, Chun J (2016) OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103.
Meier-Kolthoff JP, Göker M (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182.
Yamada K, Komagata K (1972) Transfer of Microbacterium flaccumfaciens (ex Hylemon et al. 1963) comb. nov. and Microbacterium testaceum (ex Stapp 1940) comb. nov. to the genus Curtobacterium. International Journal of Systematic Bacteriology 22 (3):335 - 345.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
21 Oct, 2025
Reviews received at journal
20 Oct, 2025
Reviewers agreed at journal
19 Oct, 2025
Reviews received at journal
17 Oct, 2025
Reviewers agreed at journal
14 Oct, 2025
Reviewers agreed at journal
13 Oct, 2025
Reviewers agreed at journal
13 Oct, 2025
Reviewers agreed at journal
01 Oct, 2025
Reviewers invited by journal
28 Aug, 2025
Editor assigned by journal
21 Aug, 2025
Submission checks completed at journal
21 Aug, 2025
First submitted to journal
20 Aug, 2025

You are reading this latest preprint version

Curtobacterium pruni sp. nov. causing black blotch on red apricot fruit in China

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Field samplings and pathogen isolation

Test of pathogenicity

Morphological Characterization

Molecular and Phylogenetic analysis

Genome sequencing and genome annotation

Results and Discussion

Field survey and pathogen isolation

Test of pathogenicity

Morphological Characterization

Molecular and Phylogenetic analysis

Genome sequencing and genome annotation

Phylogenetic analysis of the whole genome

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1